The outer membrane of Gram negative bacteria functions as a molecular sieve and allows only very small molecules to passively diffuse into the cell. Porins in the outer membrane allow the transport of larger molecules and may be specific or non-specific in their molecular recognition. Non-specific porins such as Omp F, Omp C and Pho E allow the rapid passage of hydrophilic molecules. Other porins allow the transport of specific molecules. The peptide permeases, for example, have a specificity for oligopeptides. The uptake of oligopeptides is dependent upon size, hydrophobicity and charge.
It is well documented that Escherichia coli can not take up large peptides and that the size exclusion limit for porin mediated peptide transport is 650 Daltons or the size of a penta- or hexapeptide. The size exclusion limit for peptide uptake in other Gram negative organisms such as Salmonella typhimurium has also been determined and found to be similar to that of E. coli (Payne, 1980, “Transport and utilization of peptides by bacteria,” p. 211-256. In J. W. Payne (ed.), Microorganisms and Nitrogen Sources. John Wiley & Sons, Chisester; Payne et al., 1994, Adv. Microb. Physiol. 36:1-80). In contrast to Gram negative bacteria, Gram positive bacteria can transport much larger peptides. For example, Lactococcus lactis has been shown to take up peptides over 18 residues in length or 2,140 daltons in size (Detmers et al., 1998, Biochemistry 37:16671-16679) while Bacillus megaterium can transport molecules up to 10,000 daltons in size (Scherrer et al., 1971, J. Bacteriol. 107:718-735).
Pathogenic Gram negative bacteria represent a serious threat to public health. The American Medical Association and the Centers for Disease Control and Prevention have become increasingly concerned about the dramatic increase in drug-resistance pathogens. The data below shows the incidence of Gram negative drug-resistant pathogens is the most problematic, totaling 59.9% of all drug-resistant pathogens that are monitored.
TABLE IIncidence of antimicrobial-resistant pathogensthat are monitored by the CDC.NumberPercentAntimicrobial-resistant pathogenof casesof totalMethicillin-resistant Staphylococcus aureus (MRSA)49,24714.3%Methicillin-resistant coagulase-negative29,4538.5%Staphylococci (MRCNS)Vancomycin-resistant Enterococcus spp (VRE)36,11410.5%Ceftazidime, ciprofloxacin/ofloxacin,109,16531.6%imipenem, piperacillin, or levofloxacin-resistant Pseudomonas aeruginosaCeftazidime, cefotaxime, ceftriaxone,17,2525.0%imipenem, or meropenem-resistantEnterobacter sppCeftazidime, cefotaxime, or ceftriaxone-16,8344.9%resistant Klebsiella pneumoniaeCeftazidime, cefotaxime, ceftriaxone,80,72923.4%ciprofloxacin, ofloxacin, or levofloxacin-resistant Escherichia coliCefotaxime/ceftriaxone, or penicillin-6,3281.8%resistant PnuemococciTOTAL345,122100.0%Data compiled from the CDC National Nosocomial Infections Surveillance (NNIS) August 2002 Report of Antimicrobial-Resistant Pathogens in Hospitals.
Thus, despite many medical advances, the need for antibiotics effective against Gram negative bacteria continues to increase. Unfortunately, the current size and specificity limitations on uptake of molecules by Gram negative bacteria present obstacles to the use cellular uptake machinery to deliver compounds of interest, such as antibiotics, to these pathogens. Compounding this problem is the inability of the pharmaceutical industry to readily generate new antibiotics. Pharmaceutical companies have relied on making derivatives of naturally available compounds for several decades now as evidenced by the multiple generations of new antibiotics from drug classes such as penicillins, cephalosporins, and aminoglycosides. There has been increasing interest in the development of novel peptide antibiotics, however research has focused on the development of peptide antibiotics for Gram positive pathogens due to the problem of peptide uptake by Gram negative pathogens.
Expansion of the size and type of molecules that can be taken up by Gram negative bacteria would open the door to numerous additional scientific and medical applications.